EP4004019A1 - Immunotherapy for polyomaviruses - Google Patents

Immunotherapy for polyomaviruses

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Publication number
EP4004019A1
EP4004019A1 EP20843443.1A EP20843443A EP4004019A1 EP 4004019 A1 EP4004019 A1 EP 4004019A1 EP 20843443 A EP20843443 A EP 20843443A EP 4004019 A1 EP4004019 A1 EP 4004019A1
Authority
EP
European Patent Office
Prior art keywords
epitopes
jcv
subject
peptide
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20843443.1A
Other languages
German (de)
English (en)
French (fr)
Other versions
EP4004019A4 (en
Inventor
Rajiv Khanna
George Robin Ambalathingal THOMAS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
QIMR Berghofer Medical Research Institute
Original Assignee
Queensland Institute of Medical Research QIMR
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Publication date
Application filed by Queensland Institute of Medical Research QIMR filed Critical Queensland Institute of Medical Research QIMR
Publication of EP4004019A1 publication Critical patent/EP4004019A1/en
Publication of EP4004019A4 publication Critical patent/EP4004019A4/en
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2312—Interleukin-12 (IL-12)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2315—Interleukin-15 (IL-15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
    • C12N2710/22022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
    • C12N2710/22034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Polyomaviruses are ubiquitous viruses that infect a wide range of mammalian species. Currently, more than 12 distinct human polyomavirus species have been identified, including BK polyomavirus (BKV/ Human polyomavirus 1), John Cunningham
  • JCV/ Human polyomavirus 2 JV/ Human polyomavirus 2
  • Merkel cell polyomavirus MCV/ Human polyomavirus 5
  • JCV infection may occur via the tonsils or the gastrointestinal tract and remain latent in the gastrointestinal tract, and possibly in the lymphoid organs, neuronal tissue, and kidney, where virus continues to reproduce and shed viral particles.
  • JCV and BKV may reactivate and progress to significant organ disease.
  • JC virus which can cross the blood-brain barrier into the central nervous system (CNS) where it is neurotropic, infecting glial cells (e.g .,
  • JCV infection is associated with white matter demyelination and several pathological syndromes, such as JCV granule cell layer neuronopathy (JCV GCN), JCV encephalopathy (JCV CPN/ JCVE), JCV meningitis (JCVM), and especially progressive multifocal leukoencephalopathy (PML), a
  • JCV GCN JCV granule cell layer neuronopathy
  • JCV CPN/ JCVE JCV encephalopathy
  • JCVM JCV meningitis
  • PML progressive multifocal leukoencephalopathy
  • compositions and methods related to polyomavirus epitopes that are recognized by T lymphocytes (e.g, cytotoxic T lymphocytes (CTLs) and/or helper T lymphocytes) and that are useful in the prevention and/or treatment of a polyomavirus infection (e.g, a JCV infection), and/or cancer (e.g, a polyomavirus associated cancer, such as JCV associated cancer).
  • T lymphocytes e.g, cytotoxic T lymphocytes (CTLs) and/or helper T lymphocytes
  • CTLs cytotoxic T lymphocytes
  • cancer e.g, a polyomavirus associated cancer, such as JCV associated cancer
  • the compositions and methods relate to JCV epitopes (e.g, the epitopes listed in Tables 1, 2 and 3).
  • the compositions and methods relate to hybrid epitopes that incorporate sequence variations found within a viral strain and/or across related viral strains (e.g, the epitopes listed in Table
  • a peptide e.g, an isolated and/or recombinant polypeptide comprising one or more epitopes from one or more JCV antigens ((e.g, epitopes from LTA, STA or VPl viral antigens, such as the epitopes listed in Tables 1, 2 and 3) and/or one or more hybrid epitopes (e.g, the epitopes listed in Table 4).
  • the polypeptide comprises a plurality of such epitopes.
  • the polypeptide further comprises an intervening amino acid sequence between at least two of the plurality of epitopes.
  • the peptide is capable of eliciting an immune response upon administration to a subject (e.g, a mammalian subject, such as a human subject).
  • the epitopes are selected to provide broad coverage of the human population.
  • the epitopes have HLA class I restrictions to HLA-A1, -A2, -A3, -Al l, -A23, -A24, -A26, -A29, - A30, -B7, -B8, -B27, - B35, -B38, - B40, -B41, -B44, -B51, -B56, -B57 or -B58.
  • the epitopes have HLA class II restrictions to HLA-DP, -DM, -DOA, -DOB, -DQ, or -DR.
  • the epitopes have HLA class II restrictions to HLA-DRB or -DQB.
  • the peptide comprises, consists essentially of, or consists of epitope amino acid sequences set forth in SEQ ID NOS: 1 to 21.
  • provided herein is a
  • composition comprising a peptide provided herein.
  • provided herein is a nucleic acid (e.g ., an isolated nucleic acid) encoding a peptide disclosed herein.
  • an expression construct comprising such a nucleic acid.
  • a host cell comprising such an expression construct.
  • a method of producing an isolated peptide comprising expressing the isolated peptide in the host cell of provided herein and at least partly purifying the isolated peptide.
  • a pharmaceutical composition comprising a nucleic acid provided herein.
  • a T lymphocyte e.g., a an isolated T lymphocyte, a CD4+ T lymphocyte, a CD8+ T lymphocyte
  • TCR T cell receptor
  • a method of expanding BK virus-specific T lymphocytes for adoptive immunotherapy including: (i) contacting one or more cells isolated from a subject, wherein the one or more cells comprise T lymphocytes, with an antigen presenting cell presenting an epitope provided herein; and (ii) culturing the one or more cells under conditions such that BK virus-specific T-lymphocytes are expanded from said one or more cells.
  • culturing the one or more cells is performed in the presence of IL-2 and/or IL-21.
  • the cells are cultured in the presence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 ng/ml IL-2 and/or IL-21, In some embodiments, the cells are cultured in no more than 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 ng/ml IL-2 and/or IL-21. In some embodiments, the cells are cultured in 10-50, 20-40, 25-35 or about 30 ng/ml IL-2 and/or IL-21. In some embodiments, the cells are cultured in 30 ng/ml IL-2 and/or IL-21.
  • expansion in the presence of IL-2 and/or IL-21 results in an increase in the ratio of absolute number of polyomavirus-specific CD8 T cells to the absolute number of polyomavirus-specific CD4 T cells in the expanded population of T lymphocytes.
  • a method of treating or preventing a polyomavirus infection e.g, a JCV infection
  • a polyomavirus-associated cancer e.g, a JCV-associated cancer
  • inducing a T-lymphocyte immune response in a subject comprising administering to the subject a peptide, nucleic acid, T cell or pharmaceutical composition provided herein.
  • the subject is a mammal.
  • the subject is a human.
  • the subject is immunocompromised.
  • provided herein is a method of detecting a JC virus infection in a subject, the method comprising detecting the presence of JCV-specific T lymphocytes by contacting T lymphocytes isolated from the subject with the isolated peptide provided herein. In some embodiments, the method further comprising treating the JC virus infection in the subject according to a method described herein.
  • the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is immunocompromised.
  • a cancer in a subject e.g ., a polyomavirus-associated cancer, such as a JCV- -associated cancer.
  • the method comprises administering to the subject a pharmaceutical composition comprising cytotoxic T cells (CTLs) comprising T cell receptors (TCRs) that recognize one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) of the epitopes listed in Table 1, Table 2, Table 3 and/or Table 4.
  • CTLs cytotoxic T cells
  • TCRs T cell receptors
  • the subject expresses a human leukocyte antigen (HLA) to which the one or more epitopes is restricted.
  • HLA human leukocyte antigen
  • the CTLs are autologous to the subject. In some embodiments, the CTLs are not autologous to the subject.
  • the CTLs are obtained from a CTL library or bank.
  • the method comprises administering to the subject a vaccine composition comprising one or more (e.g, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) of the epitopes listed in Table 1, Table 2, Table 3 and/or Table 4.
  • the method comprises administering to the subject a pharmaceutical composition antigen presenting cells (APCs) presenting one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
  • the subject expresses a human leukocyte antigen (HLA) to which the one or more epitopes is restricted.
  • HLA human leukocyte antigen
  • a polyomavirus infection e.g. a JCV infection
  • the subject is a polyomavirus infection
  • the method comprises administering to the subject a pharmaceutical composition comprising CTLs comprising TCRs that recognize one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20,
  • the subject expresses a HLA to which the one or more epitopes is restricted.
  • the CTLs are autologous to the subject. In some embodiments, the CTLs are not autologous to the subject. In some embodiments, the CTLs are obtained from a CTL library or bank. In some embodiments, the method comprises administering to the subject a vaccine composition comprising one or more (e.g, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18.
  • the method comprises administering to the subject a pharmaceutical composition antigen presenting cells (APCs) presenting one or more (e.g, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25,
  • the subject expresses human leukocyte antigens (HLA) to which the one or more epitopes is restricted.
  • HLA human leukocyte antigens
  • TCRs T cell receptors
  • a population of APCs presenting one or more (e.g, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) of the epitopes listed in Table 1, Table 2, Table 3 and/or Table 4.
  • the APCs comprise B cells, antigen-presenting T cells, dendritic cells and/or artificial antigen-presenting cells, such as aK562 cells.
  • the antigen-presenting cells e.g, aK562 cells
  • provided herein are methods of treating or preventing cancer (e.g, a polyomavirus associated cancer, such as a JCV associated cancer) and/or a polyomavirus (e.g., JCV) infection in a subject comprising administering the APCs described herein to a subject.
  • cancer e.g, a polyomavirus associated cancer, such as a JCV associated cancer
  • a polyomavirus e.g., JCV
  • polypeptide comprising one or more (e.g, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26,
  • nucleic acid molecule e.g ., a DNA molecule or an RNA molecule
  • polypeptide comprising one or more (e.g., at least 1, 2, 3, 4, 5,
  • the nucleic acid molecule is a vector (e.g, an adenoviral vector).
  • a vector e.g, an adenoviral vector.
  • vaccine compositions comprising a polypeptide and/or a nucleic acid molecule described herein.
  • kits for generating, activating and/or inducing proliferation of polyomavirus-specific CTLs comprising contacting CTLs with APCs that present one or more (e.g, at least 1, 2, 3, 4, 5,
  • the CTLs are contacted with APCs in vitro.
  • the APCs comprise B cells, antigen-presenting T cells, dendritic cells and/or artificial antigen- presenting cells, such as aK562 cells.
  • the antigen-presenting cells e.g, aK562 cells
  • the CTLs are contacted to the APCs in the presence of one or more cytokines.
  • provided herein are methods of generating APCs that present epitopes provided herein comprising contacting APCs with a polypeptide comprising one or more (e.g, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) of the epitopes listed in Table 1, Table 2, Table 3 and/or Table 4 and/or a nucleic acid encoding a polypeptide comprising one or more (e.g, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25,
  • the APCs express HLA to which the one or more epitopes is restricted.
  • the one or more epitopes comprise an epitope shared by two or more polyomaviruses.
  • the shared epitope comprises a region of sequence homology between the at least two polyomaviruses, and the region of sequence homology is at least 3, 4, 5, 6 or 7 amino acids across the full length of the epitope sequence.
  • the two polyomaviruses are BKV and JCV.
  • the at least three amino acids are LLL.
  • provided herein is a method of identifying a subject suitable for a method of treatment provided herein (e.g ., administration of CTLs, APCs, or vaccine compositions provided herein) comprising isolating a sample from the subject (e.g., a blood or tumor sample) and detecting the presence of an epitope provided herein, or a nucleic acid encoding an epitope provided herein.
  • the subject is identified as suitable for a method of treatment provided herein if the subject expresses an HLA to which one or more of the epitopes described herein are restricted.
  • the subject identified as being suitable for a method of treatment provided herein is treated using the method of treatment.
  • FIG. 1 shows T cell response to JCV antigens.
  • PBMCs from 17 healthy subjects were stimulated with JCV overlapping peptide pools (OPPs) and T cells expanded for 14 days in the presence of IL-2.
  • OPPs JCV overlapping peptide pools
  • the cells were assayed for the expression of IFN-g on the 14 th day upon stimulation with respective peptide pools using flow cytometry.
  • the compiled data of all 17 donors is represented in the graphs; (A) showing the response of BKV specific CD8 + T cells and (B) the response of CD4 + T cells, after stimulation with respective BKV OPPs.
  • FIG. 2 depicts representative data demonstrating the identification of T cell determinants using a two dimensional peptide matrix.
  • JCV-specific T cells expanded in vitro using the OPPs were further characterized by identifying specific T cell determinants.
  • Individual overlapping peptides were used to make subpools (24 pools; LTA1-LTA24) and the T cell response for each pool was measured by intracellular cytokine-staining (ICS) IFN-g assay as represented in the bar graph of panel A.
  • the response using the subpools were overlayed on a two dimensional matrix, thus showing the common individual peptides among the pools.
  • FACs plots of panel B demonstrate the T cell response for each individual peptide (P29, P30, and P32) when used in the IFN-g ICS assay thereby confirming the peptides responsible for eliciting JCV-specific T cell response.
  • Figure 3 shows representative data of the HLA class II restriction analysis for epitopes mapped from JCV-LT antigen. Specifically, the HLA class II-restriction of
  • VDLHAFLSQA FSNR (LT29), FLSQAVFSNRTVASF (LT30) and
  • TVASFA YTTKEKAQ (LT32) peptides as HLA DRB1*10:01 is shown. Briefly, a panel of lymphoblastoid cells (LCLs) matching one single allele of the HLA type of the donor were chosen and loaded with respective peptide for 1 hour. The loaded LCLs were then used as stimulator cells in an IFN-g ICS assay. The peptide, when presented by the MHC, elicits an IFN-g response in JCV-specific T cells.
  • LCLs lymphoblastoid cells
  • FIG. 4 demonstrates T cell cross reactivity between BKV and JCV epitopes.
  • ICS FACS plots show the expression of IFN-g in T cells expanded with either BKV epitope (SSGTQQWRGLARYFK) or JCV epitope (RSGSQQWRGLSRYFK). These primed T cells were stimulated using both BKV and JCV peptide showing that the T cells expanded with either of these epitopes recognize both BKV and JCV peptide sequences.
  • Figure 5 illustrates JCV-specific T cell expansion from healthy subjects. The frequency of CD4 + T cells expressing IFN-g was assessed and the response of each individual subject is shown in the graph.
  • Figure 6 shows the polyfunctionality of JCV-specific T cells expanded using the pooled pepdtides.
  • Representative FACs dot plots show the expression of individual effector molecules in CD4 + T cells upon re-stimulation with JCV peptide pool (a).
  • Figure 7 shows the transcription factor and effector profile of JCV-specific T cells grown in vitro.
  • compositions and methods related to polyomavirus epitopes that are recognized by T lymphocytes (e.g. , cytotoxic (CD8 + ) T lymphocytes (CTLs) and/or helper (CD4 + ) T lymphocytes) and that are useful in the prevention and/or treatment of a polyomavirus infection (e.g, a JCV infection), and/or cancer (e.g, a polyomavirus associated cancer, such as a JCV associated cancer).
  • the compositions and methods provided herein relate to JCV epitopes (e.g, the epitopes listed in Tables 1, 2 and 3).
  • the compositions and methods relate to hybrid epitopes that encompass variations found within or across BKV and JCV epitopes (e.g, the epitopes listed in Table 4).
  • administering means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering.
  • an agent can contain, for example, peptide described herein, an antigen presenting cell provided herein and/or a CTL provided herein.
  • amino acicT is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally occurring amino acids.
  • exemplary amino acids include naturally occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing.
  • binding refers to an association, which may be a stable association, between two molecules, e.g., between a TCR and a peptide/HLA, due to, for example, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions under physiological conditions.
  • tissue sample each refers to a collection of cells obtained from a tissue of a subject.
  • the source of the tissue sample may be solid tissue, as from a fresh, frozen and/or preserved organ, tissue sample, biopsy, or aspirate; blood or any blood constituents, serum, blood; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid, urine, saliva, stool, tears; or cells from any time in gestation or development of the subject.
  • cancer includes, but is not limited to, solid tumors and blood borne tumors.
  • the term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels.
  • the term“cancer” further encompasses primary and metastatic cancers.
  • homologous refers to sequence similarity (e.g, a nucleic acid or amino acid sequence) between two regions of the same sequence strand or between regions of two different sequence strands.
  • the term“ homologous” may also be used to refer to sequence similarity between two regions of the same sequence strand or between regions of two different sequence strands. For example, when an amino acid residue position in both regions is occupied by the same amino acid residue, then the regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue.
  • homology between two regions is expressed in terms of the proportion of nucleotide or amino acid residue positions of the two regions that are occupied by the same nucleotide or amino acid residue.
  • a region having the nucleotide sequence 5'-ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology.
  • the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, and preferably, at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.
  • isolated refers to material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state.
  • peptide refers to two or more amino acids linked together by peptide bonds or modified peptide bonds.
  • peptide refers to two or more amino acids linked together by peptide bonds or modified peptide bonds.
  • peptide refers to two or more amino acids linked together by peptide bonds or modified peptide bonds.
  • polypeptide refers to two or more amino acids linked together by peptide bonds or modified peptide bonds.
  • peptide refers to two or more amino acids linked together by peptide bonds or modified peptide bonds.
  • polypeptide and protein in usage herein may be used interchangeably.
  • the peptide is prepared from recombinant DNA or RNA, or of synthetic origin, or some combination thereof, which (1) is not associated with peptides that it are normally found with in nature, (2) is isolated from the cell in which it normally occurs, (3) is isolated free of other peptides from the same cellular source, (4) is expressed by a cell from a different species, or (5) does not occur in nature.
  • epitope means a peptide determinant capable of specific binding to an antibody or TCR.
  • Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains. Certain epitopes can be defined by a particular sequence of amino acids to which an antibody is capable of binding.
  • the phrase“ pharmaceutically acceptable” refers to those agents, compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • the phrase“ pharmaceutically-acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting an agent from one organ, or portion of the body, to another organ, or portion of the body.
  • a pharmaceutically-acceptable material such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting an agent from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydrox
  • polynucleotide and“ nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or
  • Polynucleotides may have any three-dimensional structure, and may perform any function.
  • polynucleotides plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present,
  • nucleotide structure may be imparted before or after assembly of the polymer.
  • a polynucleotide may be further modified, such as by conjugation with a labeling component.
  • a therapeutic that "prevents" a condition refers to a compound that, when administered to a statistical sample prior to the onset of the disorder or condition, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.
  • specific binding refers to the ability of an antibody to bind to a predetermined antigen or the ability of a peptide to bind to its predetermined binding partner.
  • an antibody or peptide specifically binds to its predetermined antigen or binding partner with an affinity corresponding to a KD of about 10 7 M or less, and binds to the predetermined antigen/binding partner with an affinity (as expressed by KD) that is at least 10 fold less, at least 100 fold less or at least 1000 fold less than its affinity for binding to a non-specific and unrelated antigen/binding partner (e.g ., BSA, casein).
  • a non-specific and unrelated antigen/binding partner e.g ., BSA, casein
  • the term“ subject” means a human or non-human animal selected for treatment or therapy.
  • therapeutically-effective amount and“ effective amount’ as used herein means the amount of an agent which, when administered to a subject, elicits adequate therapeutic response in the subject to provide beneficial outcome in the subject at a reasonable benefit/risk ratio applicable to any medical treatment.
  • Treating’ a disease in a subject or“ treating’ a subject having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug, such that at least one symptom of the disease is decreased or prevented from worsening.
  • vector refers to the means by which a nucleic acid can be propagated and/or transferred between organisms, cells, or cellular components.
  • Vectors include plasmids, viruses, bacteriophage, pro-viruses, phagemids, transposons, and artificial chromosomes, and the like, that may or may not be able to replicate autonomously or integrate into a chromosome of a host cell.
  • polyomavirus epitopes such as JCV epitopes
  • immune effector cells e.g, cytotoxic T cells/CTLs
  • the epitopes described herein are useful in the prevention and/or treatment of a polyomavirus infection (e.g ., a JCV viral infection) and/or cancer (e.g., a JVC-associated cancer expressing an epitope provided herein) and/or for the generation of pharmaceutical agents and compositions thereof (e.g, sensitized immune effector cells and/or APCs) that are useful in the prevention and/or treatment of a polyomavirus infection (e.g, JCV viral infections) and/or cancer (e.g, a polyomavirus associated cancer expressing an epitope provided herein).
  • a polyomavirus infection e.g a JCV viral infection
  • cancer e.g., a JVC-associated cancer expressing an epitope provided herein
  • pharmaceutical agents and compositions thereof e.g, sens
  • the epitope is a JCV epitope listed in Table 1, Table 2 and/or Table 3.
  • the epitope is a hybrid epitope comprising amino acids from both a BKV epitope and a homologous JCV epitope and/or amino acid variants found within different BKV or JCV strains. Exemplary hybrid epitopes are listed in Table 4.
  • the compositions and methods described herein further relate to epitopes from addition viruses, such as EB V, CMV, or ADV.
  • the epitopes are HLA class I-restricted T cell epitopes.
  • the epitopes are HLA class I-restricted T cell epitopes.
  • the epitopes are HLA class II-restricted T cell epitopes.
  • Table 2 List ofJCV epitopes and homologous BKV epitope peptide sequences
  • Table 3 Exemplary epitope sequences from JCV homologous to BKV epitope sequences
  • Table 4 Exemplary epitope sequences from JCV/BKV hybrid epitope sequences
  • peptides e.g ., polypeptides comprising one or more of the epitopes from Table 1, Table 2, Table 3 and/or Table 4.
  • the peptides disclosed herein are full-length viral proteins (e.g., full-length BKV and/or JCV proteins).
  • the peptide is not a full-length viral protein (e.g, not a full-length BKV and/or JCV protein).
  • the peptides disclosed herein comprise BKV and JCV epitopes with sequence homology (e.g, epitopes listed in Tables 2, 3 and 4).
  • the peptides disclosed herein comprise less than 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15 or 10 contiguous amino acids of a viral protein. In some embodiments, the peptides disclosed herein comprise two or more of the epitopes listed in Table 1, Table 2, Table 3 and/or Table 4. For example, in some embodiments, the peptides disclosed herein comprise two or more of the epitopes listed in Table 1, Table 2, Table 3 and/or Table 4 connected by polypeptide linkers.
  • the peptide provided herein comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 epitopes (e.g, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 of the epitopes listed in Table 1, Table 2, Table 3 and/or Table 4).
  • the peptides disclosed herein comprise the JCV epitopes set forth in Table 1, i.e., any one of the JCV epitopes set forth in SEQ ID Nos: 1-21, or any combination thereof.
  • the peptide may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
  • polypeptides e.g ., isolated polypeptides and/or recombinant polypeptides
  • polypeptides comprising a plurality of epitopes from BKV or JCV antigens (e.g., epitopes from large T-antigen (LTA), small T-antigen (STA) or major capsid protein VP1 viral antigens, such as those epitopes listed in Tables 1, 2, 3 or 4), preferably the epitopes set forth in Table 1.
  • BKV or JCV antigens e.g., epitopes from large T-antigen (LTA), small T-antigen (STA) or major capsid protein VP1 viral antigens, such as those epitopes listed in Tables 1, 2, 3 or 4
  • LTA large T-antigen
  • STA small T-antigen
  • major capsid protein VP1 viral antigens such as those epitopes listed in Tables 1, 2, 3 or 4
  • polypeptides disclosed herein comprise any one of the JCV epitopes set forth in SEQ ID Nos: 1-21, or any combination thereof.
  • the polypeptide further comprises an intervening amino acid sequence between at least two of the plurality of epitopes.
  • the intervening amino acids or amino acid sequences are proteasome liberation amino acids or amino acid sequences.
  • proteasome liberation amino acids or amino acid sequences are or comprise AD, K or R.
  • the intervening amino acids or amino acid sequence are TAP recognition motifs.
  • TAP recognition motifs may conform to the following formula:
  • TAP recognition motifs include RIW, RQW, NIW and NQY.
  • the epitopes provided herein are linked or joined by the proteasome liberation amino acid sequence and, optionally, the TAP recognition motif at the carboxyl terminus of each epitope.
  • the polypeptide comprises, or consists essentially of, each of the epitopes encoded by the amino acid sequences set forth in SEQ ID Nos: 1-21.
  • the polypeptides provided herein further comprise epitopes from and at least one additional virus (e.g, Epstein Barr virus (EBV), cytomegalovirus (CMV), and/or adenovirus (ADV)).
  • the peptides comprise epitopes two or more viruses.
  • the peptides comprise epitopes three or more viruses.
  • the peptides comprise epitopes four or more viruses.
  • the peptides comprise epitopes five or more viruses.
  • the peptides comprise sequences from at least two, three, four or five of JCV, BKV, EBV, CMV and/or ADV.
  • a polyepitope polypeptide i.e., a single chain of amino acid residues comprising multiple T cell epitopes not linked in nature
  • the T cell epitopes in the polypeptide are connected via an amino acid linker.
  • the T cell epitopes in the polypeptide are directly linked without intervening amino acids.
  • polyepitope polypeptides examples include polyepitope polypeptides, methods of generating polyepitope polypeptides, and vectors encoding poly epitope polypeptides.
  • pools of immunogenic peptides comprising HLA class I and class Il-restricted polyomavirus peptide epitopes (e.g, epitopes listed in Tables 1, 2, 3, 4, 5 and/or 6) capable of inducing proliferation of peptide-specific T cells.
  • the pool of immunogenic peptides comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 epitopes (e.g, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 of the epitopes listed in Table 1, Table 2, Table 3 and/or Table 4), or combinations thereof.
  • the peptide pool comprises at least one JCV epitope set forth in Table 1, i.e., any one of the JCV epitopes set forth in SEQ ID Nos: 1-21, or any combination thereof.
  • the pool of immunogenic peptides may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or all 21 of the epitopes encoded by the amino acid sequences set forth in SEQ ID Nos: 1-21.
  • such peptide pools comprise each of the JCV peptide epitope amino acid sequences set forth in in SEQ ID Nos: 1-21.
  • the immunogenic peptides, and pools thereof are capable of inducing proliferation of peptide-specific T cells (e.g ., peptide-specific cytotoxic T-cells and/or CD4 + T cells).
  • compositions and methods provided herein comprise or relate to naturally occurring variants of the epitopes listed in Tables 1, 2, and/or 3.
  • a polyepitope polypeptide that comprises two or more (e.g., at least 3, 4, 5, 6, 7, 8, 9 or 10) naturally occurring variants of an epitope listed in Table 1, Table 2 and/or Table 3.
  • the sequence of the epitopes provided herein have a sequence disclosed herein except for 1 or more (e.g, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) conservative sequence modifications.
  • conservative sequence modifications is intended to refer to amino acid modifications that do not significantly affect or alter the interaction between a TCR and a peptide containing the amino acid sequence presented on an HLA.
  • conservative modifications include amino acid substitutions, additions (e.g, additions of amino acids to the N or C terminus of the peptide) and deletions (e.g, deletions of amino acids from the N or C terminus of the peptide).
  • Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g ., lysine, arginine, histidine), acidic side chains (e.g, aspartic acid, glutamic acid), uncharged polar side chains (e.g, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g, threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g lysine, arginine, histidine
  • acidic side chains e.g, aspartic acid,
  • one or more amino acid residues of the peptides described herein can be replaced with other amino acid residues from the same side chain family and the altered peptide can be tested for retention of TCR binding (e.g., antigenicity) using methods known in the art.
  • Modifications can be introduced into an antibody by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • the peptides (e.g., polypeptides) described herein are immunogenic and are capable of eliciting an immune response upon administration to a subject (e.g, a mammalian subject, such as a human subject).
  • a subject e.g, a mammalian subject, such as a human subject.
  • the peptides (e.g., polypeptides) described herein are capable of eliciting an immune response following endogenous or exogenous processing and/or presentation of the peptides by immune cells (e.g., immune cells of the subject and/or immune cells from a donor such as those immune cells comprising allogeneic PBMCs.
  • cells that present one or more of the peptides described herein (e.g, a peptide comprising at least one epitope listed in Table 1, Table 2, Table 3 and/or Table 4).
  • the cell is a mammalian cell.
  • the cell is an antigen-presenting cell (APC) (e.g, an antigen-presenting T- cell, a dendritic cell, a B cell, a macrophage or am artificial antigen-presenting cell, such as aK562 cell).
  • a cell presenting a peptide described herein can be produced by standard techniques known in the art. For example, a cell may be pulsed to encourage peptide uptake.
  • the cells are transfected with a nucleic acid encoding a peptide provided herein.
  • methods of producing antigen-presenting cells comprising pulsing a cell with the peptides described herein. Exemplary examples of producing antigen-presenting cells can be found in
  • the peptides provided herein can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques, can be produced by recombinant DNA techniques, and/or can be chemically synthesized using standard peptide synthesis techniques.
  • the peptides described herein can be produced in prokaryotic or eukaryotic host cells by expression of nucleotides encoding a peptide(s) of the present invention. Alternatively, such peptides can be synthesized by chemical methods.
  • nucleic acid molecules that encode the epitopes and peptides described herein.
  • the nucleic acids may be present, for example, in whole cells, in a cell lysate, or in a partially purified or substantially pure form.
  • a nucleic acid molecule described herein can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, oligonucleotides corresponding to the nucleotide sequence of one or more of the epitopes listed in Tables 1, 2, 3, or 4 can be prepared by standard synthetic techniques, i.e., using an automated DNA synthesizer.
  • vectors e.g ., a viral vector, such as an adenovirus based expression vector
  • a viral vector may contain additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication, episomal mammalian vectors).
  • Other vectors e.g, non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes.
  • nucleic acids operable linked to one or more regulatory sequences (e.g ., a promoter) in an expression vector.
  • the cell transcribes the nucleic acid provided herein and thereby expresses an antibody, antigen binding fragment thereof, or peptide described herein.
  • the nucleic acid molecule can be integrated into the genome of the cell or it can be extrachromosomal.
  • the nucleic acid vectors or recombinant adenoviruses provided herein encode one or more epitopes listed in Tables 1, 2, 3, and/or 4.
  • the nucleic acid vectors or recombinant adenoviruses may consist of one or more epitopes from the same table (e.g., one or more epitopes from Table 1, one or more epitopes from Table 2, one or more epitopes from Table 3, or one or more epitopes from Table 4).
  • the nucleic acid vectors or recombinant adenoviruses may consist of one or more epitopes from the same table (e.g, Table 1), and one or more epitopes from a different table (e.g, Table 2).
  • the nucleic acid vectors or recombinant adenoviruses provided herein encode for no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids in addition to the epitopes listed in Tables 1, 2, 3, and/or 4.
  • the nucleic acid vectors comprise nucleic acid sequences that have undergone codon optimization.
  • a coding sequence is constructed by varying the codons in each nucleic acid used to assemble the coding sequence.
  • a method to identify a nucleotide sequence that optimizes codon usages for production of a peptide comprises at least the following steps (a) through (e).
  • step (a) oligomers are provided encoding portions of the polypeptide containing degenerate forms of the codon for an amino acid encoded in the portions, with the oligomers extended to provide flanking coding sequences with overlapping sequences.
  • step (b) the oligomers are treated to effect assembly of the coding sequence for the peptide.
  • the reassembled peptide is included in an expression system that is operably linked to control sequences to effect its expression.
  • step (c) the expression system is transfected into a culture of compatible host cells.
  • step (d) the colonies obtained from the transformed host cells are tested for levels of production of the polypeptide.
  • step (e) at least one colony with the highest or a satisfactory production of the polypeptide is obtained from the expression system.
  • the sequence of the portion of the expression system that encodes the protein is determined. Further description of codon optimization is provided in U.S. Patent
  • the HLA is a class I HLA.
  • the HLA is a class II HLA.
  • the class I HLA has an a chain polypeptide that is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-g, HLA-K or HLA-L.
  • the class II HLA has an a chain polypeptide that is HLA- DMA, HLA-DOA, HLA-DPA, HLA-DQA or HLA-DRA.
  • the class II MHLA has a b chain polypeptide that is HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB or HLA-DRB.
  • APCs present at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
  • T cell epitopes e.g, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 T cell epitopes Table 1, Table 2, Table 3 and/or Table 4).
  • the APCs are B cells, antigen presenting T-cells, dendritic cells, or artificial antigen-presenting cells (e.g, aK562 cells).
  • Dendritic cells for use in the process may be prepared by taking PBMCs from a patient sample and adhering them to plastic. Generally the monocyte population sticks and all other cells can be washed off. The adherent population is then differentiated with IL-4 and GM-CSF to produce monocyte derived dendritic cells.
  • These cells may be matured by the addition of IL-Ib, IL-6, PGE-1 and TNF-a (which upregulates the important co-stimulatory molecules on the surface of the dendritic cell) and are then contacted with a recombinant adenovirus described herein.
  • the APC is an artificial antigen-presenting cell, such as an aK562 cell.
  • the artificial antigen-presenting cells are engineered to express CD80, CD83, 41BB-L, and/or CD86.
  • Exemplary artificial antigen-presenting cells, including aK562 cells, are described U.S. Pat. Pub. No. 2003/0147869, which is hereby incorporated by reference.
  • kits for generating APCs that present the two or more of the T cell epitopes described herein comprising contacting an APC with a nucleic acid vector and/or recombinant adenoviruses encoding T cell epitopes described herein and/or with a polyepitope produced by the nucleic acid vectors or recombinant adenoviruses described herein.
  • the APCs are irradiated.
  • T cells and populations of T cells that express a TCR (e.g., an ab TCR or a gd TCR) that recognize a peptide described herein (e.g, an epitope listed in Table 1, Table 2, Table 3 and/or Table 4) presented on HLA.
  • the T cell is a CD8 T cell (a CTL) that expresses a TCR that recognizes a peptide described herein presented on a class I HLA.
  • the T cell is a CD4 T cell (a helper T cell) that recognizes a peptide described herein presented on a class II HLA.
  • the present disclosure relates to the stimulation and expansion of polyfunctional T-cells, i.e., those T cells that are capable of inducing multiple immune effector functions, that provide a more effective immune response to an epitope (e.g, an epitope listed in Table 1, Table 2, Table 3 and/or Table 4) than do cells that produce, for example, only a single immune effector (e.g, a single biomarker such as a cytokine or CD 107a).
  • a single immune effector e.g, a single biomarker such as a cytokine or CD 107a.
  • Less-polyfunctional, monofunctional, or even “exhausted” T cells may dominate immune responses during chronic infections or disease states (e.g, cancer), thus negatively impacting treatment or protection against virus- associated complications.
  • T cells may be further assessed by determining the expression patterns (e.g, expression profiles by ICS assay) of transcription factors such as T-bet and Eomes and/or cytotoxic effector molecules such as perforin and Granzyme B.
  • expression patterns e.g, expression profiles by ICS assay
  • the expression of each of T-bet, Eomes, perforin, and Granzyme B is determined for the T cells disclosed herein.
  • Such expression levels may be determined and assessed as a relative measurement such as a ratio.
  • the expression profile of T-bet / Eomes and/or Granzyme B / perforin is determined.
  • the T cells disclosed herein exhibit high expression of T-bet and low expression of Eomes (i.e., T- bet hl / Eomes l0w ); similarly, the T cells disclosed herein may exhibit high expression of Granzyme B and low expression of perforin (i.e., Granzyme 111 / Perforin low ).
  • T cells e.g., JCV-specific T cells
  • exhibiting a T-bet 111 / Eomes l0w and/or a Granzyme 111 / Perforin low expression profile are identified as functionally competent and active.
  • T cells may be selected for expansion and/or use in an adoptive T cell immunotherapy.
  • the T cells disclosed herein e.g, JCV-specific T cells
  • methods of generating, activating and/or inducing proliferation of T cells e.g ., CTLs
  • CTLs e.g ., CTLs
  • a sample comprising CTLs is incubated in culture with an APC provided herein (e.g., an APC that presents a peptide comprising a BKV and/or JCV epitope described herein on a class I HLA complex).
  • the sample containing T cells are incubated 2 or more times with APCs provided herein.
  • the T cells are incubated with the APCs in the presence of at least one cytokine.
  • the cytokine is IL-4, IL-7 and/or IL-15. Exemplary methods for inducing proliferation of T cells using APCs are provided, for example, in U.S. Pat. Pub. No. 2015/0017723, which is hereby incorporated by reference.
  • a population of CTLs collectively comprising T cell receptors that recognize one or more T cell epitopes (e.g, one or more of the T cell epitopes listed in Table 1, Table 2, Table 3 and/or Table 4).
  • the CTLs recognize two or more T cell epitopes from Table 1, Table 2, Table 3 and/or Table 4.
  • the population of CTLs collectively comprise T cell receptors that recognize T cell epitopes from any combination of JCV, BKV, EBV, CMV, ADV and/or from other viruses.
  • the population of CTLs collectively comprise T cell receptors that recognize at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38 T cell epitopes (e.g, at least 1, 2, 3, 4, 5, 6, or 7 T cell epitopes from Table 1 and/or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of the epitopes listed in Table 1, Table 2, Table 3 and/or Table 4).
  • T cell receptors that recognize at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38 T cell epitopes (e.g, at least 1, 2, 3, 4, 5, 6, or 7 T cell epitopes from Table 1 and/or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
  • provided herein are methods of preventing or treating a
  • compositions comprising the nucleic acid vector described herein, peptides produced by the nucleic acid vector described herein, CTLs and/or APCs provided herein (e.g, comprising the nucleic acid vector described herein) and a pharmaceutically acceptable carrier.
  • the CTLs and/or APCs are not autologous to the subject (i.e., the CTLs and/or APCs are allogeneic to the subject).
  • the T cells and/or APCs are autologous to the subject.
  • the T cells and/or APCs are stored in a cell bank before they are administered to the subject.
  • a composition e.g ., a pharmaceutical composition, such as a vaccine composition
  • a peptide e.g., comprising an epitope from Table 1
  • nucleic acid e.g., nucleic acid vector, recombinant adenovirus, antibody, CTL, or an APC described herein formulated together with a pharmaceutically acceptable carrier, as well as methods of treating cancer (e.g., a polyomavirus associated cancer, such as a JVC associated cancer) or a polyomavirus infection (e.g, a JCV, CMV, EBV, or ADV infection) using such pharmaceutical compositions.
  • the composition includes a combination of multiple (e.g, two or more) agents provided herein.
  • the pharmaceutical composition further comprises an adjuvant.
  • adjuvant broadly refers to an agent that affects an immunological or physiological response in a patient or subject.
  • an adjuvant might increase the presence of an antigen over time or to an area of interest like a tumor, help absorb an antigen-presenting cell antigen, activate macrophages and lymphocytes and support the production of cytokines.
  • an adjuvant might permit a smaller dose of an immune interacting agent to increase the effectiveness or safety of a particular dose of the immune interacting agent.
  • an adjuvant might prevent T cell exhaustion and thus increase the effectiveness or safety of a particular immune interacting agent.
  • adjuvants include, but are not limited to, an immune modulatory protein, Adjuvant 65, a-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, b-Glucan Peptide, CpG oligodeoxynucleotides, non-CpG
  • oligodeoxynucleotides e.g., GPI-0100, lipid A and modified versions thereof (e.g,
  • lipid A monophosphorylated lipid A
  • lipopolysaccharide lipovant
  • Montanide Montanide
  • N-acetyl- muramyl-L-alanyl-D-isoglutamine Pam3CSK4
  • quil A quil A
  • TLR9 agonist ODNla
  • ODNla a cationic antimicrobial peptide (CAMP) such as KLK, IC31, and trehalose dimycolate.
  • CAMP cationic antimicrobial peptide
  • Methods of preparing these formulations or compositions include bringing into association an agent described herein with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association an agent described herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • compositions of this invention suitable for parenteral administration comprise one or more agents described herein in combination with one or more
  • sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • the agents of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
  • JCV sequences and/or protein expression can be observed in several malignancies, and frequently reported in immunocompromised (e.g ., immunodeficient, immuno- incompetent, and/or immunosuppressed) patients with and without PML. Accordingly, in certain aspects, provided herein are methods of treating and/or preventing cancer (e.g., a polyomavirus-associated cancer, such as a JCV-associated cancer) or a polyomavirus infection (e.g., a JCV infection). In some embodiments, the method comprises
  • composition comprising a CTL, APC, polypeptide and/or nucleic acid molecule described herein.
  • the subject treated is immunocompromised.
  • the subject has a T cell deficiency.
  • the subject has leukemia, lymphoma (e.g, Hodgkin’s lymphoma) or multiple myeloma.
  • the subject has multiple sclerosis, psoriasis, and/or other autoimmune diseases.
  • the subject is infected with HIV and/or has AIDS.
  • the subject has undergone a tissue, organ and/or bone marrow transplant.
  • the subject is receiving immunosuppressive therapy such as steroids, cytostatics and antiproliferative agents, therapeutic antibodies, calcineurin inhibitors, anti rejection drugs, and the like or combinations thereof.
  • immunosuppressive therapy such as steroids, cytostatics and antiproliferative agents, therapeutic antibodies, calcineurin inhibitors, anti rejection drugs, and the like or combinations thereof.
  • the subject has undergone and/or is undergoing chemotherapy. In some embodiments, the subject has undergone and/or is undergoing radiation therapy.
  • the subject is suffering from a JCV infection in the central nervous system (e.g. re-activation of a JCV infection or seeding of newly reactivated virus).
  • the JCV infection is associated with destruction of oligodendrocytes and/or white matter demyelination.
  • the subject is suffering from JCV granule cell layer neuronopathy (JCV GCN), JCV encephalopathy (JCV CPN/ JCVE), JCV meningitis (JCVM), and/or progressive multifocal leukoencephalopathy (PML), preferably from PML.
  • the pathogen e.g., JCV
  • JCV is detectable in the cerebrospinal fluid of the subject.
  • the subject has cancer.
  • the methods described herein may be used to treat any cancerous or pre-cancerous tumor.
  • the cancer expresses one or more of the polyomavirus epitopes provided herein (e.g, the BKV/JCV epitopes listed in Tables 1, 2, 3, and/or 4). In some
  • the cancer is a JVC -associated carcinoma.
  • the cancer includes a solid tumor.
  • the cancer is a gastrointestinal malignancy, such as colon cancer, gastric cancer, gastrointestinal tumors, and the like.
  • the cancer is a CNS malignancy, such as gliomas and all subtypes thereof (e.g, ependymomas, astrocytomas, brainstem gliomas, oligodendrogliomas, optic nerve gliomas, mixed gliomas, and the like), medulloblastomas, primitive neuroectodemal tumors, and neuroblastomas.
  • cancers that may be treated by methods and compositions provided herein includecancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma;
  • transitional cell carcinoma papillary transitional cell carcinoma; adenocarcinoma;
  • gastrinoma malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma;
  • nonencapsulating sclerosing carcinoma adrenal cortical carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous
  • adenocarcinoma adenocarcinoma
  • ceruminous adenocarcinoma adenocarcinoma
  • mucoepidermoid carcinoma adenocarcinoma
  • cystadenocarcinoma papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous iadenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; mammary paget's disease; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma; malignant granulosa cell tumor; and malignant roblastoma; sertoli cell carcinoma; malignant leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extra-mammary paraganglioma; pheochromocytoma;
  • glomangiosarcoma malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma;
  • myxosarcoma liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal
  • rhabdomyosarcoma alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma;
  • malignant mesenchymoma malignant brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant struma ovarii; choriocarcinoma; malignant mesonephroma;
  • hemangiosarcoma malignant hemangioendothelioma; kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; malignant odontogenic tumor; ameloblastic
  • odontosarcoma malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; glioblastoma multiforme; pineoblastoma, gliosarcoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemmoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hod
  • the subject is also administered an anti -viral drug that inhibits polyomavirus replication.
  • an anti -viral drug that inhibits polyomavirus replication.
  • the subject is administered ganciclovir, valganciclovir, foscarnet, cidofovir, acyclovir, formivirsen, maribavir, BAY 38-4766 or GW275175X.
  • the subject is also administered an immune checkpoint inhibitor.
  • Immune Checkpoint inhibition broadly refers to inhibiting the checkpoints that cancer cells can produce to prevent or downregulate an immune response.
  • immune checkpoint proteins include, but are not limited to, CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, TIM-3 or VISTA.
  • Immune checkpoint inhibitors can be antibodies or antigen binding fragments thereof that bind to and inhibit an immune checkpoint protein.
  • immune checkpoint inhibitors include, but are not limited to, atezolizumab, avelumab, camrelizumab, cemiplimab, cetrelimab, durvalumab (MEDI-4736), genolimzumab, ipilimumab, nivolumab, pembrolizumab, pidilizumab, sintilimab, spartalizumab, tislelizumab, Toripalimab, AMP-224, AMP-514, AK-104, ASP- 8374, AUR-012, BCD-135, BGB-A333, BMS-936559, CBT-502, MCLA-145, KN-046, MGD-019, MK-4830, MSB-0020718C, RG-7446, SL-279252, STI-A1010, STI-A1110, TSR-042, XmAb20717, and XmAb23104.
  • composition provided herein is administered
  • compositions may be administered prior to or after the detection of cancer cells or polyoma virus-infected cells in a subject.
  • a composition provided herein is administered prior to or after the administration of an immunosuppressive therapy (e.g, steroids, cytostatics and
  • the composition is administered prior to or after chemotherapy.
  • the composition is administered prior to or after radiation therapy.
  • a proinflammatory response is induced.
  • the proinflammatory immune response comprises production of proinflammatory cytokines and/or chemokines, for example, interferon gamma (IFN-g) and/or interleukin 2 (IL-2).
  • Conjunctive therapy includes sequential, simultaneous and separate, and/or co administration of the active compounds in such a way that the therapeutic effects of the first agent administered have not entirely disappeared when the subsequent treatment is administered.
  • the second agent may be co-formulated with the first agent or be formulated in a separate pharmaceutical composition.
  • provided herein is a method of identifying a subject suitable for a therapy provided herein (e.g ., methods of treating a polyoma virus infection, such as JCV infection, and/or cancer in a subject comprising administering to the subject a
  • the method comprises isolating a sample from the subject (e.g., a blood sample, a tissue sample, a tumor sample) and detecting the presence of an epitope listed in Tables 1, 2 or 3 in the sample.
  • the epitope is detected using an ELISA assay, a western blot assay, a FACS assay, a fluorescent microscopy assay, an Edman degradation assay and/or a mass spectrometry assay (e.g., protein sequencing).
  • the presence of a JCV epitope for example, is detected by detecting a nucleic acid encoding the JCV epitope.
  • the nucleic acid encoding the JCV epitope is detected using a nucleic acid probe, a nucleic acid amplification assay and/or a sequencing assay.
  • the JC viral genome is composed of two conserved coding regions separated by a highly variable non-coding control region (NCCR) harboring both sequences required for replication (ORI, the origin of viral replication) and for transcription (several promoters and c/.s-regulating elements); a highly conserved region which includes ORI is followed by sections a, b, c, d, e and f.
  • NCCR highly variable non-coding control region
  • JC virus found in the CNS of PML patients is often found to have rearranged NCCRs (e.g, absence of b and d sections and duplication of the a-c-e sequence). Differences in NCCR sequence may contribute to the fitness of the virus in the CNS and thus to the development of PML. Accordingly, provided herein are methods of identifying a subject suitable for a therapy provided herein, comprising isolating a sample from the subject (e.g ., a blood sample, a urine sample, a tissue sample, a cerebrospinal fluid sample, a tumor sample) and detecting the presence of PML-associated JCV sequence
  • nucleic acid amplification techniques such as nested PCR and the like.
  • sequences and methods of detection are known in the art, such as in
  • the method comprises HLA typing of the subject.
  • the subject is identified as suitable for treatment with a method provided herein if the subject expresses an HLA to which an epitope provided herein is restricted.
  • the methods provided herein further comprise treating the identified subject using a therapeutic method provided herein (e.g., by administering to the subject a pharmaceutical composition provided herein).
  • the subject is administered a composition comprising CTLs described herein, wherein the CTLs comprise TCRs that recognize an epitope provided herein that is HLA restricted to an HLA expressed by the subject.
  • the subject is administered a composition comprising a polypeptide comprising an epitope provided herein that is HLA restricted to an HLA expressed by the subject.
  • the subject is administered a composition comprising an APC presenting a polypeptide comprising an epitope provided herein that is HLA restricted to an HLA expressed by the subject.
  • the subject is administered a composition comprising an nucleic acid encoding a polypeptide comprising an epitope provided herein that is HLA restricted to an HLA expressed by the subject.
  • Example 1 CD8 + and CD4 + T cell responses directed towards LTA, VP1 and STA JCV antisens
  • PBMCs from 17 healthy volunteers were incubated with JVC overlapping peptide pools (OPPs) and cultured for 14 days in the presence of IL-2.
  • Peptide matrices for each of large T antigen (LTA), small T antigen (STA), and viral protein 1 (VP1), as well as the composition of the peptide pools for each matrix, were arranged as follows.
  • T cell cultures were assessed for JVC-specificity using an intracellular cytokine (ICS) assay.
  • ICS intracellular cytokine
  • peptide 32 P32
  • FACS Fluorescence-activated cell sorting
  • Example 3 JCV -specific T cell expansion and characterization.
  • JCV -specific T cells were expanded in vitro following stimulation with pooled JCV epitopes. Specifically, PBMC from healthy volunteers were stimulated with synthetic JCV peptides (Table 1) for 1 hour and then cultured for 12-14 days in the presence of different cytokine combinations, including IL-2 (lOng/ml), IL7 (lOng/ml), IL12 (lOng/ml) and/or IL15 (lOng/ml). The JCV specificity of the expanded T cells was assessed using standard intracellular cytokine assays (Table 6). Table 6: CD4 + T cell Responses to JCV Peptides
  • Example 4 T cell cross-reactivity between JCV and BKV epitopes.
  • Peptide-specific T cells were expanded in vitro following stimulation with JCV epitope or BKV epitope and then re-stimulated with the corresponding homologous peptide epitope (see Table 2) so as to observe any cross-reactive response between JCV and BKV epitopes.
  • PBMC from healthy volunteers were cultured with synthetic JCV peptide epitope RSGSQQWRGLSRYFK or with synthetic BKV peptide epitope
  • T cells expanded with either of the homolgous epitopes recognize both the BKV and JCV peptide sequence.
  • Example 5 Profiling functional and phenotypic characteristics of JCV-specific T cells in healthy individuals and transplant recipients
  • T-box transcription factors T-bet
  • Eomes Eomesodermin
  • T-bet, Eomes, perforin and granzyme B are assayed on JCV-specific T cells and CMV specific T cells using ICS.
  • Initial analysis shows a medium to low level of T bet expression in JCV-specific T cells while high levels of T-bet are seen with CMV specific T cells.
  • Very low expression of Eomes is found with JCV-specific T cells in comparison to the CMV specific T cells.
  • low levels of perforin and granzyme B are also seen with JCV-specific T cells.
  • PBMCs from 15 healthy donors were selected randomly irrespective of their HLA type and JCV-specific T cells were expanded upon stimulation with a pool of peptides comprising the peptides disclosed herein (i.e ., peptides comprising the amino acid sequences set forth in SEQ ID NOs. 1-21).
  • the T cells were expanded for 17 days and assessed for JCV response using intracellular cytokine staining assay.
  • T cells from thirteen out of fifteen donors had JCV-specific T cell response which is evidenced by the production of IFN-g upon re- stimulation with the peptide pool (see Figure 5).
  • JCV-specific T cells were analysed for their expression of IL-2, TNF, IFN-g and CD 107 by intracellular staining (see Figure 6a). JCV-specific T cells showed a higher expression of TNF along with other cytokines.

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